This application is a competing renewal of a very successful project entitled "Cyclic Amplification of Prion Protein Misfolding". In the short period of 4 years, this project has produced some major contributions to the field, which is clearly reflected by the number and quality of the publications produced (19 research articles, some published in the most prestigious scientific journals, such as Cell, Science, Nature Medicine, EMBO J, Nature Biotech, PLoS Pathogen, JBC, among others). Prion diseases are a group of infectious neurodegenerative disorders associated with the cerebral accumulation of misfolded protein aggregates. Although rare diseases, the recent outbreak of Bovine Spongiform Encephalopathy and Chronic Wasting disease and the transmission of the disease from cattle to humans have risen a great public health concern. This problem is aggravated by many uncertainties surrounding the unprecedented nature of the infectious agent, its mechanism of propagation and the species-barrier that seems to control prion transmission. The most accepted hypothesis proposes that the infectious agent (termed prion) is composed exclusively of a misfolded version of a normal protein and does not contain any nucleic acid. According to this hypothesis, the disease is transmitted by propagation of the misfolding from the disease associated isoform (termed PrPSc) to the normal host protein (termed PrPC). We have described a procedure to replicate prions in vitro with high efficiency. This technology, named Protein Misfolding Cyclic Amplification (PMCA) mimics the process of prion replication in vivo, but at an accelerated speed resulting in an exponential amplification of the initial amount of PrPSc. PMCA studies have provided important contributions to understand the nature of the infectious agent, the mechanism of replication and to detect minute quantities of prions with high efficiency and sensitivity. The major goal of this project is to take advantage of the PMCA technology to generate infectious material from purified recombinant prion protein and to understand the molecular basis of three unique phenomena associated to prions: prion strains, species barrier and prion memory. This project offers to continue a very successful path of research to address some of the key pending questions in the prion field that are essential to minimize further transmission of prion diseases and the emergence of novel forms of prions with potentially devastating consequences. PUBLIC HEALTH RELEVANCE: Prions are the heretical proteinaceous infectious agents responsible for a group of human and animal diseases. This project proposes to answer some of the most intriguing scientific questions surrounding the unprecedented nature of prions, including the generation of infectivity from synthetic prion protein, the mechanism of prion strain diversity, species barrier and the phenomenon of prion molecular memory. Answers to these questions not only will provide exciting scientific advances, but will also contribute to minimize further spreading of prions and the future emergence of novel prion forms with potentially devastating consequences for human health.